1. Field of the Invention
The present invention relates to microemulsion formulations of free-form and/or conjugation-stabilized therapeutic agents, and to methods of making and using same. The compositions of the invention may comprise therapeutic agents such as proteins, peptides, nucleosides, nucleotides, antiviral agents, antineoplastic agents, antibiotics, antiarrhythmics, anti-coagulants, etc., and prodrugs, precursors, derivatives, and intermediates thereof.
2. Description of the Related Art
In the field of pharmaceutical therapeutic intervention, and the treatment of disease states and physiological conditions, a wide variety of therapeutic agents have come into use, including various proteins, peptides, nucleosides, nucleotides, antiviral agents, antineoplastic agents, antibiotics, antiarrhythmics, anti-coagulants, etc., and prodrugs precursors, derivatives, and intermediates of the foregoing.
For example, the use of polypeptides and proteins for the systemic treatment of specific diseases is now well accepted in medical practice. The role that the peptides play in replacement therapy is so important that many research activities are being directed towards the synthesis of large quantities by recombinant DNA technology. Many of these polypeptides are endogenous molecules which are very potent and specific in eliciting their biological actions. Other non-(poly)peptidyl therapeutic agents are equally important and pharmaceutically efficacious.
A major factor limiting the usefulness of these therapeutic substances for their intended application is that they are easily metabolized by plasma proteases when given parenterally. The oral route of administration of these substances is even more problematic because in addition to proteolysis in the stomach, the high acidity of the stomach destroys them before they reach their intended target tissue. For example, polypeptides and protein fragments, produced by the action of gastric and pancreatic enzymes, are cleaved by exo and endopeptidases in the intestinal brush border membrane to yield di- and tripeptides, and even if proteolysis by pancreatic enzymes is avoided, polypeptides are subject to degradation by brush border peptidases. Any of the therapeutic agent that survives passage through the stomach is further subjected to metabolism in the intestinal mucosa where a penetration barrier prevents entry into the cells.
In spite of these obstacles, there is substantial evidence in the literature to suggest that nutritional and pharmaceutical therapeutic agents such as proteins are absorbed through the intestinal mucosa. On the other hand, nutritional and drug (poly)peptides are absorbed by specific peptide transporters in the intestinal mucosa cells. These findings indicate that properly formulated therapeutic agents such as (poly)peptides and proteins may be administered by the oral route, with retention of sufficient biological activity for their intended use. If, however, it were possible to modify these therapeutic agents so that their physiological activities were maintained totally, or at least to a significant degree, and at the same time stabilize them against proteolytic enzymes and enhance their penetration capability through the intestinal mucosa, then it would be possible to utilize them properly for their intended purpose. The product so obtained would offer advantages in that more efficient absorption would result, with the concomitant ability to use lower doses to elicit the optimum therapeutic effect.
The problems associated with oral or parenteral administration of therapeutic agents such as proteins are well known in the pharmaceutical industry, and various strategies are being used in attempts to solve them. These strategies include incorporation of penetration enhancers, such as the salicylates, lipid-bile salt-mixed micelles, glycerides, and acylcarnitines, but these frequently are found to cause serious local toxicity problems, such as local irritation and toxicity, complete abrasion of the epithelial layer and inflammation of tissue. These problems arise because enhancers are usually co-administered with the therapeutic agent and leakages from the dosage form often occur. Other strategies to improve oral delivery include mixing the therapeutic agent with protease inhibitors, such as aprotinin, soybean trypsin inhibitor, and amastatin, in an attempt to limit degradation of the administered therapeutic agent. Unfortunately these protease inhibitors are not selective, and endogenous proteins are also inhibited. This effect is undesirable.
Enhanced penetration of therapeutic agents across mucosal membranes has also been pursued by modifying the physicochemical properties of candidate drugs. Results indicate that simply raising lipophilicity is not sufficient to increase paracellular or transcellular transport. Indeed it has been suggested that cleaving peptide-water hydrogen bonds is the main energy barrier to overcome in obtaining diffusion of peptide therapeutics across membranes (Conradi, R. A., Hilgers, A. R., Ho, N. F. H., and Burton, P. S., "The influence of peptide structure on transport across Caco-2 cells", Pharm. Res., 8 1453-1460, (1991)). Protein stabilization has been described by several authors. Abuchowski and Davis ("Soluble polymers-Enzyme adducts", In: Enzymes as Drugs, Eds. Holcenberg and Roberts, J. Wiley and Sons, New York, N.Y., (1981)) disclosed various methods of derivatization of enzymes to provide water soluble, non-immunogenic, in vivo stabilized products.
A great deal of work dealing with protein stabilization has been published. Abuchowski and Davis disclose various ways of conjugating enzymes with polymeric materials (Ibid.). More specifically, these polymers are dextrans, polyvinyl pyrrolidones, glycopeptides, polyethylene glycol and polyamino acids. The resulting conjugated polypeptides are reported to retain their biological activities and solubility in water for parenteral applications. The same authors, in U.S. Pat. No. 4,179,337 (issued Dec. 18, 1979), disclose that polyethylene glycol rendered proteins soluble and non-immunogenic when coupled to such proteins. These polymeric materials, however, did not contain fragments suited for intestinal mucosa binding, nor did they contain any moieties that would facilitate or enhance membrane penetration. While these conjugates were water-soluble, they were not intended for oral administration.
Meisner et al., U.S. Pat. No. 4,585,754 Apr. 29, 1986, teaches that proteins may be stabilized by conjugating them with chondroitin sulfates. Products of this combination are usually polyanionic, very hydrophilic, and lack cell penetration capability. They are usually not intended for oral administration.
Mill et al., U.S. Pat. No. 4,003,792 (issued Oct. 16, 1990) teaches that certain acidic polysaccharides, such as pectin, algesic acid, hyaluronic acid and carrageenan, can be coupled to proteins to produce both soluble and insoluble products. Such polysaccharides are polyanionic, derived from food plants. They lack cell penetration capability and are usually not intended for oral administration.
In Pharmacological Research Communication 14, 11-120 (1982), Boccu et al. disclosed that polyethylene glycol could be linked to a protein such as superoxide dismutase ("SOD"). The resulting conjugated product showed increased stability against denaturation and enzymatic digestion. The polymers did not contain moieties that are necessary for membrane interaction and thus suffer from the same problems as noted above in that they are not suitable for oral administration.
Other techniques of stabilizing peptide and protein drugs in which proteinaceous drug substances are conjugated with relatively low molecular weight compounds such as aminolethicin, fatty acids, vitamin B.sub.12, and glycosides, are described in the following articles: R. Igarishi et al., "Proceed. Intern. Symp. Control. Rel. Bioact. Materials, 17, 366, (1990); T. Taniguchi et al. Ibid 19, 104, (1992); G. J. Russel-Jones, Ibid, 19, 102, (1992); M. Baudys et al., Ibid, 19, 210, (1992).
The modifying compounds are not polymers and accordingly do not contain moieties necessary to impart both the solubility and membrane affinity necessary for bioavailability following oral as well as parenteral administration. Many of these preparations lack oral bioavailability.
Another approach which has been taken to lengthen the in vivo duration of action of proteinaceous substances is the technique of encapsulation. M. Saffran et al., in Science, 223, 1081, (1986) teaches the encapsulation of proteinaceous drugs in an azopolymer film for oral administration. The film is reported to survive digestion in the stomach but is degraded by microflora in the large intestine, where the encapsulated protein is released. The technique utilizes a physical mixture and does not facilitate the absorption of released protein across the membrane.
Ecanow, U.S. Pat. No. 4,963,367 (issued Oct. 16, 1990), teaches that physiologically active compounds, including proteins, can be encapsulated by a coacervative-derived film and the finished product can be suitable for transmucosal administration. Other formulations of the same invention may be administered by inhalation, oral, parenteral and transdermal routes. These approaches do not provide intact stability against acidity and proteolytic enzymes of the gastrointestinal tract, the property as desired for oral delivery.
Another approach taken to stabilize protein drugs for oral as well as parenteral administration involves entrapment of the therapeutic agent in liposomes. A review of this technique is found in Y. W. Chien, "New Drug Delivery Systems", Marcel Dekker, New York, N.Y., 1992. Liposome-protein complexes are physical mixtures; their administration gives erratic and unpredictable results. Undesirable accumulation of the protein component in certain organs has been reported, in the use of such liposome-protein complexes. In addition to these factors, there are additional drawbacks associated with the use of liposomes, such as cost, difficult manufacturing processes requiring complex lypophilization cycles, and solvent incompatibilities. Moreover, altered biodistribution and antigenicity issues have been raised as limiting factors in the development of clinically useful liposomal formulations.
The use of "proteinoids" has been described recently (Santiago, N., Milstein, S. J., Rivera, T., Garcia, E., Chang., T. C., Baughman, R. A., and Bucher, D., "Oral Immunization of Rats with Influenza Virus M Protein (M1) Microspheres", Abstract #A 221, Proc. Int. Symp. Control. Rel. Bioac. Mater., 19, 116 (1992)). Oral delivery of several classes of therapeutics has been reported using this system, which encapsulates the drug of interest in a polymeric sheath composed of highly branched amino acids. As is the case with liposomes, the drugs are not chemically bound to the proteinoid sphere, and leakage of drug out of the dosage form components is possible.
A peptide which has been the focus of much synthesis work, and efforts to improve its administration and bioassimilation, is insulin.
The use of insulin as a treatment for diabetes dates back to 1922, when Banting et al. ("Pancreatic Extracts in the Treatment of Diabetes Mellitus," Can. Med. Assoc. J., 12, 141-146 (1922)) showed that the active extract from the pancreas had therapeutic effects in diabetic dogs. Treatment of a diabetic patient in that same year with pancreatic extracts resulted in a dramatic, life-saving clinical improvement. A course of daily injections of insulin is required for extended recovery.
The insulin molecule consists of two chains of amino acids linked by disulfide bonds; the molecular weight of insulin is around 6,000. The .beta.-cells of the pancreatic islets secrete a single chain precursor of insulin, known as proinsulin. Proteolysis of proinsulin results in removal of four basic amino acids (numbers 31, 32, 64 and 65 in the proinsulin chain: Arg, Arg, Lys, Arg respectively) and the connecting ("C") peptide. In the resulting two-chain insulin molecule, the A chain has glycine at the amino terminus, and the B chain has phenylalanine at the amino terminus.
Insulin may exist as a monomer, dimer or a hexamer formed from three of the dimers. The hexamer is coordinated with two Zn.sup.2+ atoms. Biological activity resides in the monomer. Although until recently bovine and porcine insulin were used almost exclusively to treat diabetes in humans, numerous variations in insulin between species are known. Porcine insulin is most similar to human insulin, from which it differs only in having an alanine rather than threonine residue at the B-chain C-terminus. Despite these differences most mammalian insulin has comparable specific activity. Until recently animal extracts provided all insulin used for treatment of the disease. The advent of recombinant technology allows commercial scale manufacture of human insulin (e.g., Humulin.TM. insulin, commercially available from Eli Lilly and Company, Indianapolis, Ind.).
Although insulin has now been used for more than 70 years as a treatment for diabetes, few studies of its formulation stability appeared until two recent publications (Brange, J., Langkjaer, L., Havelund, S., and V.o slashed.lund, A., "Chemical stability of insulin. I. Degradation during storage of pharmaceutical preparations," Pharm. Res., 2, 715-726, (1992); and Brange, J. Havelund, S., and Hougaard, P., "Chemical stability of insulin. 2. Formulation of higher molecular weight transformation products during storage of pharmaceutical preparations," Pharm. Res., 9, 727-734, (1992)). In these publications, the authors exhaustively describe chemical stability of several insulin preparations under varied temperature and pH conditions. Earlier reports focused almost entirely on biological potency as a measure of insulin formulation stability. However the advent of several new and powerful analytical techniques--disc electrophoresis, size exclusion chromatography, and HPLC--allows a detailed examination of insulin's chemical stability profile. Early chemical studies on insulin stability were difficult because the recrystallized insulin under examination was found to be no more than 80-90% pure. More recently monocomponent, high-purity insulin has become available. This monocomponent insulin contains impurities at levels undetectable by current analysis techniques.
Formulated insulin is prone to numerous types of degradation. Nonenzymatic deamidation occurs when a side-chain amide group from a glutaminyl or asparaginyl residue is hydrolyzed to a free carboxylic acid. There are six possible sites for such deainidation in insulin: Gln.sup.A5, Gin.sup.A15, Asn.sup.A18, Asn.sup.A21, Asn.sup.B3, and Gln.sup.B4. Published reports suggest that the three Asn residues are most susceptible to such reactions.
Brange et al. (ibid) reported that in acidic conditions insulin is rapidly degraded by extensive deamidation at Asn.sup.A21. In contrast, in neutral formulations deamidation takes place at Asn.sup.B3 at a much slower rate, independent of insulin concentration and species of origin of the insulin. However, temperature and formulation type play an important role in determining the rate of hydrolysis at B3. For example, hydrolysis at B3 is minimal if the insulin is crystalline as opposed to amorphous. Apparently the reduced flexibility (tertiary structure) in the crystalline form slows the reaction rate. Stabilizing the tertiary structure by incorporating phenol into neutral formulations results in reduced rates of deamidation.
In addition to hydrolytic degradation products in insulin formulations, high molecular weight transformation products are also formed. Brange et al. showed by size exclusion chromatography that the main products formed on storage of insulin formulations between 4 and 45.degree. C. are covalent insulin dimers. In formulations containing protamine, covalent insulin protamine products are also formed. The rate of formulation of insulin-dimer and insulin-protamine products is affected significantly by temperature. For human or porcine insulin, (regular N1 preparation) time to formation of 1% high molecular weight products is decreased from 154 months to 1.7 months at 37.degree. C. compared to 4.degree. C. For zinc suspension preparations of porcine insulin, the same transformation would require 357 months at 4.degree. C. but only 0.6 months at 37.degree. C.
These types of degradation in insulin may be of great significance to diabetic subjects. Although the formation of high molecular weight products is generally slower than the formation of hydrolytic (chemical) degradation products described earlier, the implications may be more serious. There is significant evidence that the incidence of immunological responses to insulin may result from the presence of covalent aggregates of insulin (Robbins, D. C. Cooper, S. M. Fineberg, S. E., and Mead, P. M., "Antibodies to covalent aggregates of insulin in blood of insulin-using diabetic patients", Diabetes, 36, 838-841, (1987); Maislos, M., Mead, P. M., Gaynor, D. H., and Robbins, D. C., "The source of the circulating aggregate of insulin in type I diabetic patients is therapeutic insulin", J. Clin. Invest., 77, 717-723. (1986); and Ratner R. E., Phillips, T. M., and Steiner, M., "Persistent cutaneous insulin allergy resulting from high molecular weight insulin aggregates", Diabetes, 39, 728-733, (1990)). As many as 30% of diabetic subjects receiving insulin show specific antibodies to covalent insulin dimers. At a level as low as 2% it was reported that the presence of covalent insulin dimers generated a highly significant response in lymphocyte stimulation in allergic patients. Responses were not significant when dimer content was in the range 0.3-0.6%. As a result it is recommended that the level of covalent insulin dimers present in formulation be kept below 1% to avoid clinical manifestations.
Several insulin formulations are commercially available; although stability has been improved to the extent that it is no longer necessary to refrigerate all formulations, there remains a need for insulin formulations with enhanced stability. A modified insulin which is not prone to formation of high molecular weight products would be a substantial advance in the pharmaceutical and medical arts, and modifications providing this stability (and in addition providing the possibility of oral availability of insulin) would make a significant contribution to the management of diabetes.
In addition to the in vivo usage of therapeutic agents including polypeptides, proteins, nucleosides, and other biologically active molecules, such agents also find substantial and increasing use in diagnostic reagent applications. In many such applications, these agents are utilized in solution environments wherein they are susceptible to thermal and enzymic degradation. Examples of such diagnostic agents include enzymes, peptide and protein hormones, antibodies, enzyme-protein conjugates used for immunoassay, antibody-hapten conjugates, viral proteins such as those used in a large number of assay methodologies for the diagnosis or screening of diseases such as AIDS, hepatitis, and rubella, peptide and protein growth factors used for example in tissue culture, enzymes used in clinical chemistry, and insoluble enzymes such as those used in the food industry. As a further specific example, alkaline phosphatase is widely utilized as a reagent in kits used for the colorimetric detection of antibody or antigen in biological fluids. Although such enzyme is commercially available in various forms, including free enzyme and antibody conjugates, its storage stability in solution often is limited. As a result, alkaline phosphatase conjugates are frequently freeze-dried, and additives such as bovine serum albumin and Tween 20 are used to extend the stability of the enzyme preparations. Such approaches, while advantageous in some instances to enhance the resistance to degradation of the therapeutic and/or diagnostic agents, have various shortcomings which limit their general applicability.
In general, the approaches of the prior art for formulating proteinaceous therapeutic agents for enhanced stability in vivo do not provide intact stability of such agents against acid and proteolytic enzymes of the gastrointestinal tract, the property desired for oral delivery of protein drugs. In spite of this fact, intensive efforts are being made in pharmaceutical and scientific organizations toward oral administration of protein drugs. These efforts have largely focused on insulin as a protein drug of choice for developing oral dosage forms, but have not been successful in yielding formulations that replace parenteral administration.
Formulations of free insulin using different techniques have been attempted.
Liposome entrapped insulin for oral administration and its attendant drawbacks have been described hereinabove.
A recent approach involves formulation of insulin in a liquid medium, using absorption enhancers. U.S. Pat. No. 5,653,987 (issued Aug. 5, 1997) to Modi and Chandarana teaches that insulin can be formulated for oral or nasal delivery using at least two different absorption enhancers. A close examination of the enhancers described in this reference reveals that these enhancers are not pharmaceutically acceptable. In the examples provided in this patent, most contain either sodium lauryl sulphate, a detergent known to damage the lining of the gastrointestinal tract, or polyoxyethylene 9-lauryl ether, which is used in extracting protein from biological specimens, and additionally is known to be spermatocidal in character. The formulation and synthesis method of U.S. Pat. No. 5,653,987 (issued Aug. 5, 1997) differ fundamentally from the concept and method of the present invention. Additionally, unlike the present invention, the formulation approach of U.S. Pat. No. 5,653,987 (issued Aug. 5, 1997) does not address enzymatically stabilized insulin conjugates.
Still another recent approach of free insulin formulation for oral delivery utilizes the technique of microemulsion. This formulation approach has been described by Cho, Y. W., et al, in U.S. Pat. Nos. 5,656,289 (issued Aug. 12, 1997) and 5,665,700 (issued Sep. 9, 1997), by Owen, Albert J., in U.S. Pat. No. 5,646,109 (issued Jul. 8, 1997) and by Desai, Ashok J., in U.S. Pat. No. 5,206,219 (issued Apr. 27, 1993).
Cho, Y. W., et al teaches in U.S. Pat. No. 5,656,289 (issued Aug. 12, 1997) that oral proteinaceous compositions comprising oil/water emulsions can form chylomicra or provide chylomicra to sites of absorption in the gastrointestinal tract. Such patent teaches that the hydrophilic phase may contain ethanol. In the disclosed examples of microemulsion preparations, a substantial amount of ethanol is needed in the hydrophilic portion of the emulsion. Ethanol, however, is known to denature many proteinaceous drugs. Further, the method of manufacturing the proteinaceous composition described by Cho et al. in U.S. Pat. No. 5,656,289 (issued Aug. 12, 1997) involves microfluidization, which can damage or denature protein drugs as a result of the heat generation and shear force entailed in the microfluidization process.
In U.S. Pat. No. 5,665,700 (issued Sep. 9, 1997), Cho et al. teach that proteinaceous compounds can be orally delivered in a water-in-oil formulation comprising a hydrophilic phase dispersed in a lipophilic phase to form an emulsion. Microfluidization with its aforementioned attendant drawbacks is also disclosed in U.S. Pat. No. 5,665,700 (issued Sep. 9, 1997) for preparing the microemulsion. Free insulin (insulin that is not modified by conjugation with amphiphilic polymers, as disclosed hereinafter and in U.S. Pat. No. 5,681,811 issued Oct. 28, 1997, U.S. Pat. No. 5,438,040 issued Aug. 1, 1995 and U.S. Pat. No. 5,259,030 issued Oct. 25, 1994, all in the name of Nnochiri Nkem Ekwuribe) is unstable in the gastrointestinal tract. In the examples provided in U.S. Pat. No. 5,665,700 (issued Sep. 9, 1997), most preparations disclosed by Cho et al. contain protease inhibitors. Inhibition in such formulations will, however, not be specific to insulin, and inhibitors may cause severe gastrointestinal problems as a result of inhibition of intestinal proteinaceous contents which are otherwise digestible. Further, lecithin complexation with insulin is required in Cho et al.'s microemulsion formulation. In the present invention, the use of lecithin is not essential.
As a result of the protease inhibition resulting from Cho et al.'s formulations containing protease inhibitors, indiscriminate absorption of toxic proteinaceous material may result in the in vivo use of such formulations. The formulations of the Cho et al. U.S. Pat. Nos. 5,656,289 (issued Aug. 12, 1997) and 5,665,700 (issued Sep. 9, 1997) are based on a concept and a method of manufacture that differ fundamentally from the concept and method of the herein disclosed invention.
Another microemulsion formulation of free insulin is described in U.S. Pat. No. 5,646,109 (issued Jul. 8, 1997) to A. J. Owen. Owen teaches that a water-in-oil (w/o) microemulsion readily converts to an oil-in-water (o/w) emulsion by the addition of aqueous fluid to the w/o microemulsion. A close examination of the conditions necessary for convertible microemulsion in the formulation of U.S. Pat. No. 5,646,109 (issued Jul. 8, 1997) reveals that the resultant HLB (hydrophilic and lipophilic balance required is greater than 7. In one example described at column 22, lines 26-28 of U.S. Pat. No. 5,646,109 (issued Jul. 8, 1997), Owen demonstrates that his formulation containing resultant HLB of 4 produces a non-convertible microemulsion and has no effect in promoting rectal delivery of calcitonin. The use of resultant HLB of greater than 7 to prepare a convertible microemulsion for oral delivery of insulin is based on a concept that differs from the concept of herein disclosed invention, as hereinafter more fully described.
Desai, A. J., U.S. Pat. No. 5,206,219 (issued Apr. 27, 1993) describes another microemulsion formulation for oral administration, in which a liquid polyol solvent and lipid cosolvent containing a proteinaceous medicament, e.g., insulin, is treated to form a microemulsion in the gastrointestinal tract at sites of absorption. Desai teaches that a vital ingredient in the formulation is a protease inhibitor. The purpose of using the inhibitor is to prevent the degradation of the proteinaceous medicament. Problems that arise from using protease inhibitors to aid in oral delivery of proteinaceous drugs have been enumerated hereinabove.
International Publications WO 93-02664 (issued February, 1993) and WO94-08610 (issued Apr. 28, 1994) of Constantides et al. describe compositions of w/o microemulsion formulations containing peptides and proteins using medium chain fatty acid triglycerides, surfactants and a hydrophilic phase containing proteins. The ratio of triglycerides to low HLB surfactants in these preparations can be 5:1 to 1.5:1. Constantides et al. also describe the preparation of microemulsion compositions containing salts of medium chain fatty acids (high HLB surfactant) in which the insulin loading is 0.35 mg/mL. Contrary to the approach of the invention hereinafter disclosed, a careful examination of the examples of the Constantides et al., International Publications WO 93-02664 (issued February, 1993) and WO94-08610 (issued Apr. 28, 1994) reveals their combined HLB strength to be above 7.
It would therefore be a substantial advance in the art, and is correspondingly an object of the present invention, to provide an improved formulation for the administration of therapeutic agents such as proteins, peptides, nucleosides, nucleotides, antiviral agents, antineoplastic agents, antibiotics, antiarrhythmics, anti-coagulants, etc., and prodrugs precursors, derivatives, and intermediates of the foregoing, which avoids the foregoing problems.
It is another object of the invention to provide an improved formulation for the oral administration of protein and peptide therapeutic agents, in which the therapeutic agent is stabilized against proteolysis and degradation in the gastrointestinal tract.
It is yet another object of the invention to provide an improved formulation for administration of therapeutic agents which are of free form and/or are conjugatively stabilized as described in U.S. Pat. No. 5,681,811 issued Oct. 28, 1997, U.S. Pat. No. 5,438,040 issued Aug. 1, 1995 and U.S. Pat. No. 5,259,030 issued Oct. 25, 1994, all in the name of Nnochiri Nkem Ekwuribe.
Other objects and advantages of the present invention will be more fully apparent from the ensuing disclosure and appended claims.